CN101942407B - Producing strain and production method for producing microbial flocculant from wheat starch waste water - Google Patents
Producing strain and production method for producing microbial flocculant from wheat starch waste water Download PDFInfo
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- CN101942407B CN101942407B CN2010102598736A CN201010259873A CN101942407B CN 101942407 B CN101942407 B CN 101942407B CN 2010102598736 A CN2010102598736 A CN 2010102598736A CN 201010259873 A CN201010259873 A CN 201010259873A CN 101942407 B CN101942407 B CN 101942407B
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Abstract
The invention relates to a producing strain for producing microbial flocculant from wheat starch waste water and a production method thereof. The producing strain is Bacillus sp.-TY35 which is preserved in China Center for Type Culture Collection with the preservation number of CCTCC NO: M2010166. The production method comprises the following steps: the culture of a seed liquid, the fermentation and the production of the flocculant, and the extraction of the flocculant. By using low-cost starch wastewater as the culture medium to prepare the microbial flocculant, the producing strain effectively uses the wheat starch waste water, saves the carbon source cost in the traditional culture medium, and greatly reduces the production cost of the microbial flocculant. The production method for producing the microbial flocculant has the advantages of high yield, short flocculant-producing cycle and wide application range, and has favorable turbidity removal and COD removal effects on the wastewater.
Description
Technical field
The invention belongs to the effluent sewage treatment and disposal technical field in the environmental engineering, relate to the production preparation of microbial flocculant, specifically is a kind of production bacterium and working method thereof of utilizing wheat starch waste water to produce microbial flocculant.
Background technology
At present; The treatment process of both at home and abroad food and wastewater from fermentation industry and sewage being used always can be divided into biological method, physical method and chemical process generally; Small particles of pollution thing to colloidal attitude, ultra colloidal attitude and the suspended state that can not remove with the natural sedimentation method in the waste water; Generally it is taken off surely, be gathered into and remove behind the sedimentary oarse-grained alumen ustum through adding flocculation agent.Flocculation agent can significantly improve sedimentation speed and treatment effect, and flocculation sedimentation is widely used as a kind of lower-cost water treatment method, in water treatment, occupies extremely important status.The quality of flocculation sedimentation treatment effect depends on the performance of flocculation agent to a great extent; Present flocculation agent comprises inorganic salts (Tai-Ace S 150, aluminum chloride, iron(ic)chloride), high molecular polymerization inorganic salts (Poly aluminum Chloride (PAC), poly-ferric chloride) and high score organic polymer (SEPIGEL 305) product, and they have obtained good effect in water treatment.But the use meeting of above-mentioned various flocculation agents causes secondary pollution to environment, can work the mischief to human health like aluminum ion, and SEPIGEL 305 is difficult to degraded, and the monomer whose acrylic amide has stronger carcinogenesis.Therefore the flocculation agent of efficient, the safety non-toxic of market active demand, environmental protection.
Microbial flocculant (Microbial flocculants; MBF) be one type of biomacromolecule with throwing out by microorganisms, it is compared with traditional flocculation agent, has following unique advantage: (1) is prone to be degraded by microorganisms; Nontoxic, safe; (2) non-secondary pollution; (3) use range is wide, and is effective; (4) have turbidity removal and decoloration performance; (5) have also have the pH of receiving value influence little, Heat stability is good, characteristics such as consumption is little.The staple of microbial flocculant has gp, polysaccharide, protein, Mierocrystalline cellulose and DNA etc., and it has makes the sedimentary performance of other coagulation of materials.Microbial flocculant generally obtains through microbial fermentation, extracting, refinings such as bacterium, fungi, yeast.Up to now, found that the product MBF types of spawn that screens comprises mould, bacterium, actinomycetes, yeast etc.
Though microbial flocculant possesses above-mentioned advantage, because its culture medium cost is higher, combined coefficient is low, and the microbial flocculant cost is high, and the research of most of microbe flocculation agent still is in the laboratory development.Therefore,, can effectively solve the too high problem of culture medium cost, thereby the potential business opportunity is provided as substratum producing microbial in next life flocculation agent with cheap carbon source for commercially producing of flocculation agent.In recent years, the report of this respect is arranged successively, and studied penicillium spp HHE-P7 produces microbial flocculant in sauce waste water flocculating properties like Liu Hui etc., optimum flocculating condition is for to add 10-15mL in 1L kaolin water.Dong Shuanshi etc. are substratum with the waste liquid of fermentation method biological hydrogen production reactor discharging, have investigated the optimal conditions of fermentation that composite efficient flcos producing bacteria F2-F6 produces biological flocculant.
Summary of the invention
The objective of the invention is to overcome the deficiency that exists in the prior art, a kind of production bacterium that utilizes wheat starch waste water to produce microbial flocculant is provided, this production bacterium adopts cheap starch wastewater to prepare microbial flocculant as substituting substratum; The present invention simultaneously also provides the fermentation method for producing of said microbial flocculant.
First purpose of the present invention solves through following technical scheme: utilize wheat starch waste water to produce the production bacterium of microbial flocculant; It is characterized in that: this production bacterium is genus bacillus-TY35 (Bacillussp.-TY35); It has been preserved in Chinese typical culture collection center, and deposit number is CCTCC NO:M2010166.
Second purpose of the present invention solves through following technical scheme: utilize wheat starch waste water to produce the working method of microbial flocculant, comprise the fermentative prodn of culture of seed liquid, flocculation agent and the extraction of flocculation agent, it is characterized in that:
(1), culture of seed liquid: produce bacterium (in the seed culture fluid after genus bacillus-TY35) is seeded to autoclaving through the microbial flocculant of acclimation shaking culture; Shaking table is cultivated 10～20h under 20～40 ℃ of conditions; Shaking speed is 180r/min, to bacterial concentration be 1.0～5.0 * 10
8CFU/mL;
(2), the fermentative prodn of flocculation agent: get an amount of wheat starch waste water; Be adjusted to 9000mg/L≤CODcr≤30000mg/L and (, then add appropriate amount of starch, like CODcr＞30000mg/L like CODcr＜9000mg/L; Thin up then); Ratio according to 1～3g/L is added steeping water, regulates pH to 6.0～7.0 (ammoniacal liquor is simultaneously as replenishing nitrogenous source) with ammoniacal liquor then, is mixed with liquid fermentation medium; In liquid fermentation medium, inoculate the seed liquor of 2～10% volume ratios, then at 100-250r/min, ventilating fermentation is cultivated 40～50h under 35 ℃ the condition;
(3), the extraction of flocculation agent: with fermented liquid centrifugal 10～20min under the condition of 5000～8000r/min of step (2) acquisition; Collect supernatant, add 3-4 absolute ethyl alcohol doubly by volume, stirring and placing under 4 ℃ staticly settles 12h; Centrifugal under 4000～6000r/min condition then; Abandon supernatant, collecting precipitation and vacuum-drying obtain solid microbe flocculation agent bullion to constant weight.
Said seed culture fluid is: get an amount of wheat starch waste water, in wheat starch waste water, add steeping water according to the ratio of 10～20g/L, regulate pH to 6～7 with ammoniacal liquor then, at 121 ℃ of sterilization 20min.
The present invention compared with prior art, advantage is:
(1), the substratum of producing microbial flocculant production among the present invention is that the starch wastewater with high density is a carbon source; Add a spot of steeping water and less ammonia; Ammoniacal liquor had not only been regulated the pH value but also as replenishing nitrogenous source; Effectively utilize wheat starch waste water, saved the carbon source cost in traditional substratum, made the production cost of microbial flocculant reduce greatly.
(2), to utilize working method of the present invention to produce the output of microbial flocculant high, it is short, applied widely to produce the wadding cycle, turbidity removal, the COD of waste water is removed have good effect.
(3), the present invention had both reduced the production cost of microbial flocculant; Solve simultaneously the reluctant problem of high density wheat starch waste water again; Realized waste water reclaiming; This bacterial classification can be used for fields such as microbial water-purifying agent, environmental microorganism preparation simultaneously, has broad application prospects, and is fit to large-scale industrial production and popularization.
Embodiment
The screening and separating process that microbial flocculant of the present invention is produced bacterium is following: the starch wastewater mud sample is cultivated with 30 ℃ electro-heating standing-temperature cultivator culture plates through dilution, enrichment culture, and single bacterium colony is obtained in line repeatedly; To vigorous, the more sticking single bacterium colony of growth on the screening culture medium flat board, the separation of repeatedly ruling again is until using microscopy to confirm that thalline is purer single bacterium colony; The bacterial strain that utilizes flocculating effect to test primary dcreening operation carries out multiple sieve, obtains bacterial strain at last.It is yellow slightly partially that this bacterial strain is grown to white at broth culture, irregular bacterium colony, toughness, starch capable of using; Hydrolyzed casein is shaft-like through microscopy, produces gemma; Preliminary evaluation is a bacillus, and it has been preserved in Chinese typical culture collection center, and deposit number is CCTCC NO:M2010166.
Below in conjunction with specific embodiment the present invention is described further.
The preservation of culture samples of the present invention:
Bacterial classification: genus bacillus-TY35 (Bacillus sp.-TY35); Preservation date: culture samples is preserved in Chinese typical culture collection center on July 1st, 2010, and deposit number is CCTCC NO:M2010166; Preservation address: Chinese Wuhan Wuhan University.
The equipment that the present invention uses: the mechanical agitating fermentation tank of 5L (the moist biotechnology equipment ltd in Nanjing produces); Whizzer (Anting Scientific Instrument Factory, Shanghai makes, model TGL-16C); Vacuum drying oven (Shanghai Yiheng Scientific Instruments Co., Ltd, model BPH-6063); Ultraviolet-visible pectrophotometer (UNICO(Shanghai) Instruments Co., Ltd. produces, model UV-2800AH).
Embodiment 1
Wheat starch waste water is taken from Jiangsu Lianhai Biotechology Co., Ltd., and its CODcr is 9620mg/L, and pH is 4.25.
(1), seed liquor is cultivated:
(a) seed culture fluid: get an amount of wheat starch waste water, in wheat starch waste water, add steeping water, regulate pH to 6.5 with ammoniacal liquor then, at 121 ℃ of sterilization 20min according to the ratio of 10g/L;
Culture condition: (genus bacillus-TY35), be inoculated in the seed culture fluid is cultivated 20h to the production bacterium of picking slant preservation under 35 ℃ of conditions, shaking speed 180r/min is about 1.0 * 10 to bacterial concentration
8CFU/mL is with this seed liquor as when inoculation;
(2), the fermentative prodn of flocculation agent:
(a) liquid fermentation medium: 5 liters of fermentor tanks, the 3.5 liters of wheat starch waste water of packing into, in wheat starch waste water, add steeping water according to the ratio of 1.1g/L, regulate pH to 6.5 with ammoniacal liquor then, at 121 ℃ of sterilization 20min;
(b) inoculate the seed liquor of 5% volume ratio to liquid fermentation medium, then at 200r/min, ventilating fermentation is cultivated 48h under 35 ℃ the condition, and ventilation is 0.4-1.0v/v/m;
(3), the extraction of flocculation agent: get the fermented liquid that 100mL step (2) obtains, centrifugal 10min under the condition of 8000r/min collects supernatant, obtains liquid flocculation agent bullion; The absolute ethyl alcohol that adds 4 times by volume, stirring and placing under 4 ℃ staticly settles 12h, centrifugal under the 4000r/min condition then; Abandon supernatant; Collecting precipitation and vacuum-drying obtain solid microbe flocculation agent bullion 0.64g to constant weight, and flocculation agent content is 6.4g/L in the fermented liquid.
(4), kaolin floc test:
(a) be in the Kaolin clay suspension of 4g/L in 80mL concentration, add 5mL 1%CaCl
2Solution, 2mL liquid flocculation agent bullion, adding distil water is to 100mL again; Regulate pH value to 7.0; Pour in the 150mL beaker, be placed on the magnetic stirring apparatus and stir 3min fast, leave standstill 5min after; Draw the supernatant of certain depths with suction pipe, under the wavelength of 550nm, measure absorbance A with ultraviolet-visible pectrophotometer;
(b) replace 2ml liquid flocculation agent bullion with 2ml zero(ppm) water simultaneously, carry out above-mentioned same operation, record absorbance A 0;
(c) calculate flocculating rate according to formula E=(A0-A)/A0 * 100%.In the formula: the E-flocculating rate, unit is %; A0-does not have the optical density(OD) of flocculation treatment; Optical density(OD) after the A-flocculation treatment.
Through measuring test-results: after adding the processing of liquid flocculation agent bullion in the Kaolin clay suspension, flocculating rate reached 94%.This liquid flocculation agent bullion is after boiling water boils 5 minutes, and adopting and measuring flocculating rate with quadrat method is 91.4%, and the thermostability of the microbial flocculant that visible the present invention obtains is stronger.
Embodiment 2
Wheat starch waste water COD cr is 18500mg/L, and the pH value is 4.10.
Wheat starch waste water is taken from Jiangsu Lianhai Biotechology Co., Ltd., and its CODcr is 18500mg/L, and pH is 4.10.
(1), seed liquor is cultivated:
(a) seed culture fluid: get an amount of wheat starch waste water, in wheat starch waste water, add steeping water, regulate pH to 6.5 with ammoniacal liquor then, at 121 ℃ of sterilization 20min according to the ratio of 15g/L;
Culture condition: (genus bacillus-TY35), be inoculated in the seed culture fluid at 36 ℃, is cultivated 15h under the shaking speed 180r/min condition to the production bacterium of picking slant preservation, is about 2 * 10 to bacterial concentration
8CFU/mL is with this seed liquor as when inoculation;
(2), the fermentative prodn of flocculation agent:
(a) liquid fermentation medium: 5 liters of fermentor tanks, the 3.5 liters of wheat starch waste water of packing into, in wheat starch waste water, add steeping water according to the ratio of 2.1g/L, regulate pH to 6.5 with ammoniacal liquor then, at 121 ℃ of sterilization 20min;
(b) inoculate the seed liquor of 8% volume ratio to liquid fermentation medium, then at 250r/min, ventilating fermentation is cultivated 42h under 35 ℃ the condition, and ventilation is 0.4-1.0v/v/m;
(3), the extraction of flocculation agent: get the fermented liquid that 100mL step (2) obtains, centrifugal 15min under the condition of 7000r/min collects supernatant, obtains liquid flocculation agent bullion; The absolute ethyl alcohol that adds 3.5 times by volume, stirring and placing under 4 ℃ staticly settles 12h, centrifugal under the 5000r/min condition then; Abandon supernatant; Collecting precipitation and vacuum-drying obtain solid microbe flocculation agent bullion 1.04g to constant weight, and flocculation agent content is 10.4g/L in the fermented liquid.
(4), kaolin floc test:
TP is with embodiment 1, through measuring test-results: the flocculating rate reached 96.4% of the Kaolin clay suspension after the adding liquid flocculation agent bullion flocculation treatment.
Embodiment 3
Wheat starch waste water is taken from Jiangsu Lianhai Biotechology Co., Ltd., and its CODcr is 28500mg/L, and the pH value is 4.0.
(1), seed liquor is cultivated:
(a) seed culture fluid: get an amount of wheat starch waste water, in wheat starch waste water, add steeping water, regulate pH to 6.5 with ammoniacal liquor then, at 121 ℃ of sterilization 20min according to the ratio of 20g/L;
Culture condition: (genus bacillus-TY35), be inoculated in the seed culture fluid at 36 ℃, is cultivated 14h under the shaking speed 180r/min condition to the production bacterium of picking slant preservation, is about 5 * 10 to bacterial concentration
8CFU/mL is with this seed liquor as when inoculation;
(2), the fermentative prodn of flocculation agent:
(a) liquid fermentation medium: 5 liters of fermentor tanks, the 3.0 liters of wheat starch waste water of packing into, in wheat starch waste water, add steeping water according to the ratio of 3.0g/L, regulate pH to 6.5 with ammoniacal liquor then, at 121 ℃ of sterilization 20min;
(b) inoculate the seed liquor of 6% volume ratio to liquid fermentation medium, then at 200r/min, ventilating fermentation is cultivated 45h under 35 ℃ the condition, and ventilation is 0.4-1.0v/v/m;
(3), the extraction of flocculation agent: get the fermented liquid that 100mL step (2) obtains, centrifugal 20min under the condition of 6000r/min collects supernatant, obtains liquid flocculation agent bullion; The absolute ethyl alcohol that adds 3 times by volume, stirring and placing under 4 ℃ staticly settles 12h, centrifugal under the 6000r/min condition then; Abandon supernatant; Collecting precipitation and vacuum-drying obtain solid microbe flocculation agent bullion 1.26g to constant weight, and flocculation agent content is 12.6g/L in the fermented liquid.
(4), kaolin floc test:
TP is with embodiment 1, through measuring test-results: the flocculating rate reached 97.2% of the Kaolin clay suspension after the adding liquid flocculation agent bullion flocculation treatment.
Embodiment 4: simultaneous test
Select for use the microbial flocculant of embodiment 3 preparations respectively municipal effluent (taking from Wuxi north of the city sewage work), diet waste water (taking from Southern Yangtze University's the 3rd dining room water drain) and waste water from dyestuff (taking from Nanjing, Zhangjiagang Fabritex S.r.l. dyeing waste water) to be carried out flocculation treatment; Promptly in the various waste water of 100mL, add the liquid flocculation agent bullion 2mL of embodiment 3 preparations respectively; Measure flocculating rate and CODcr clearance then, the result is as shown in table 1:
Table 1 microbial flocculant is to the flocculating effect of actual waste water
Can find out that from table 1 microbial flocculant of embodiment 3 preparations all has good flocculation turbidity removal effect to three kinds of actual waste water.Observe in the experiment, microbial flocculant is handled 5min and can be formed big floc sedimentation and sinking rapidly the best results of municipal effluent, and water quality is as clear as crystal behind the 30min, and flocculating rate can be up to 93.1%.
Claims (3)
1. utilize wheat starch waste water to produce the production bacterium of microbial flocculant; It is characterized in that: this production bacterium is genus bacillus-TY35 (Bacillus sp.-TY35); It has been preserved in Chinese typical culture collection center, and deposit number is CCTCC NO:M2010166.
2. utilize wheat starch waste water to produce the working method of microbial flocculant, comprise the fermentative prodn of culture of seed liquid, flocculation agent and the extraction of flocculation agent, it is characterized in that:
(1), culture of seed liquid: producing bacterium through the microbial flocculant of acclimation shaking culture is in the seed culture fluid after genus bacillus-TY35 (Bacillus sp.-TY35) is seeded to autoclaving; Shaking table is cultivated 10～20h under 20～40 ℃ of conditions; Shaking speed is 180r/min, to bacterial concentration be 1.0～5.0 * 10
8CFU/mL;
(2), the fermentative prodn of flocculation agent: get an amount of wheat starch waste water; Be adjusted to 9000mg/L≤CODcr≤30000mg/L, add steeping water, regulate pH to 6.0～7.0 with ammoniacal liquor then according to the ratio of 1～3g/L; Ammoniacal liquor as replenishing nitrogenous source, is mixed with liquid fermentation medium simultaneously; In liquid fermentation medium, inoculate the seed liquor of 2～10% volume ratios, then at 100-250r/min, ventilating fermentation is cultivated 40～50h under 35 ℃ the condition;
(3), the extraction of flocculation agent: with fermented liquid centrifugal 10～20min under the condition of 5000～8000r/min of step (2) acquisition; Collect supernatant, add 3-4 absolute ethyl alcohol doubly by volume, stirring and placing under 4 ℃ staticly settles 12h; Centrifugal under 4000～6000r/min condition then; Abandon supernatant, collecting precipitation and vacuum-drying obtain solid microbe flocculation agent bullion to constant weight.
3. the working method of microbial flocculant as claimed in claim 2; It is characterized in that: said seed culture fluid is: get an amount of wheat starch waste water; Ratio according to 10～20g/L is added steeping water in wheat starch waste water, regulate pH to 6～7 with ammoniacal liquor then, at 121 ℃ of sterilization 20min.
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| CN102925489A (en) * | 2012-10-10 | 2013-02-13 | 临安天川环保科技有限公司 | Preparation method of microbial flocculant |
| CN106566792B (en) * | 2016-11-14 | 2019-08-20 | 江苏师范大学 | A kind of method and bacterial strain uses therefor of directly conversion rice chaff production microbial flocculant |
| CN112125383B (en) * | 2020-07-23 | 2024-04-16 | 信阳师范学院 | Method for preparing biological flocculant by using glutinous rice processing wastewater |
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| CN100491522C (en) * | 2006-04-11 | 2009-05-27 | 湖南大学 | Bacteria for producing microorganism flocculant by using bean dregs and process for producing same |
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